This thesis presents a study into the use of microgels to restore degenerated intervertebral discs (IVDs). This was undertaken using a pH-triggered microgel which was able to form self-supporting gels through an increase in pH. The microgels were based on the poly(A/MAA/X) formulation, where MAA is methacrylic acid and A is the structural and X is the crosslinking monomer. The microgel particles were also used to construct composite poly(ethylene glycol) dimethacrylate (PEGD) hydrogels. The properties of the microgels and the composite hydrogels were then investigated. Microgel particles were synthesised based on poly(EA/MAA/BDD) (poly(ethyl acrylate/MAA/butanediol diacrylate). These were able to swell on increasing pH. Concentrated dispersions formed a gel with a high elastic modulus. The EA and BDD were replaced with related monomers and gave gels with different properties. Using monomers with high glass transition temperatures reduced the rate of swelling, and using monomers with similar reactivity ratios appeared to produce more uniformly crosslinked particles. It is proposed from the data presented that those with a large difference in reactivity ratio resulted in microgel particles with a change in crosslinking gradient through the radius of the particle. In some cases this produced microgels which appeared to fragment on increasing pH. The microgels investigated were based on poly(EA/MAA/X), (E-X) with BDD, EGD (ethylene glycol dimethacrylate) and PEGD. The EGD and PEGD microgels were shown to fragment with increasing pH. Poly(EA/MAA/PEGD) dispersions were able to form a gel at a pH below the pKa which appeared to be an electrostatically repulsive gel. Following this work, it became apparent that the E-BDD microgel was the most ideal of all the microgels with gels giving low values tanδ and frequency dependence of tanδ (tanδ = G"/G', where G" is the viscous modulus and G' is the elastic modulus). It also appeared to give physical gels with the highest elastic modulus. This microgel was therefore used for the composite gels. The poly(EA/MAA/BDD) microgel was then used to form covalently-linked composite hydrogels with PEGD of different molecular weights. PEGD with a molecular weight less than 550 formed a hydrogel-linked microgel, with interpenetrating polymer chains. These composites had high G' values and swelling ratios. Using PEGD with molecular weights higher than 550 produced microgel-filled hydrogels which had high values for G' and swelling ratios. Furthermore, due to osmotic deswelling of the microgel particles, the dispersions underwent a gel-to-fluid transition prior to being heated with initiator and crosslinked. This meant that some of the formulations were injectable. The mixture of high molecular weight PEGD and microgel was therefore combined with an accelerator which enabled gel formation and crosslinking at physiological temperature. Composites formed under physiological conditions were then tested for their ability to support biomechanically meaningful loads using degenerated IVDs. The discs were then compressed and the compressive strain to measured. The results showed that the composite was able to restore the mechanical modulus and height of the degenerated disc, showing favourable results for future research.

In order to adopt an efficient bipedal posture and method of locomotion, the human skeleton has evolved a curved vertebral column and a stable, compact male pelvic girdle. Adaptive vertebral curves and the force of gravity render it susceptible to injury and degenerative change. This study examines if there is any association between pelvic size and shape and the distribution, type and severity of vertebral degenerative disease. Four documented North- west European, middle-class skeletal samples from the eighteenth and nineteenth century were examined. Pelvic shape and size were recorded, the latter of which necessitated the measurementof 93 dimensions. The severity and distribution of osteophytes, porosity and eburnation in the vertebral column were recorded. Statistical analysis was undertaken of relationships between pelvic measurements and tile sex and age at death of individuals as well as correlations between the measurements themselves. The relationship between pelvic shape and degenerative disease was also investigated. The correlation between measurements in the pelvis and disease were examined and a mechanism was created to display this relationship. Results demonstrated that this sample exhibited significant din-iorphic differcrices in pelvic measurements and pelvic shape between the sexes. Significant correlations were found between age and pelvic dirriensions in five (331/o)sacral, 29 (94%) innominate and four (25%) reconstructed pelvis measurements. Correlations were small but positive for both sexes in the sacrum and innorninate. In the reconstructed pelvis, significant correlations were again small, but Positive in females and negative in males, suggesting that although a larger pelvis may be selected for in older females, the opposite is occurring with males. This supports the theory of an evolutionary effect selecting for females with larger pelves and males with more compact pelves. Statistical analyses of tile relationship between pelvic shape and the severity or presence/absence of degenerative disease were limited and not deemed to have any statistical ment. A 'signpost' configuration was created to graphically display results of correlations between individual measurements and disease. Results suggest that osteophytosis was the most common type of disease encountered and the superior and inferior body surfaces were the main areas affected, particularly in the lower half of the thoracic and lumbar regions. All, correlations, except one, were positive, implying a positive association between those measurements and the degree of degenerative change. Patterning of the correlations was identified and discussed and statistical differences between correlations at levels of maximum and minimum curvature were examined. Results suggest that particular pelvimetry plays a significant role in this at levels of maximum and minimuni curvature. Discriminant function analysis was employed to explore the predictive ability of combinations of measurements to predispose to the development and severity of osteophytosis on the superior vertebral body surface. Contrived data was then used to test this model and was successful in predicting an expected level of expression of pathological change.

Idiopathic scoliosis (lS) is a structural lateral curvature of the spine with rotation for which no cause is established. Surgical treatment for scoliosis focuses on the spine and achieves only partial correction of spine and trunk deformity. This correction deteriorates with time. Some pathomechanisms of deteriorating body shape are suggested from sequential anthropometry. The correction and prevention of future deterioration in body shape are the aims of any scoliosis treatment. Application of knowledge of pathomechanisms to treatment may improve outcome. Section 1: Infantile and juvenile idiopathic scoliosis: long-term follow-up and effects of Luque trolley instrumentation and anterior release and convex epiphysiodesis. Patients with infantile (IIS) and juvenile idiopathic scoliosis (JIS) are evaluated by radiological examination before surgery and at intervals after surgery. The patients are also reviewed clinically at longest follow-up by surface and ultrasound methods. Appropriate non-parametric and parametric tests and multivariate analysis are used to evaluate results. Factors important in curve progression are identified and new strategies for treatment suggested. Section 2: Adolescent Idiopathic Scoliosis: 2 year follow-up and effects of each of posterior and anterior instrumentation with the Universal Spine System. Patients with adolescent idiopathic scoliosis (AIS) are evaluated before surgery and at intervals after surgery. Data from surface, anthropometry, questionnaire and plain radiography are considered. Statistical analyses were performed using parametric and nonparametric tests where appropriate. Attention is directed at factors that determine rib-hump progression post-operatively. Aims of studies: The aims of these studies are to quantitate the change in surface and skeletal morphology after surgery and after follow-up, to infer pathogenesis and pathomechanisms for each of infantile and adolescent idiopathic scoliosis, to consider new strategies for the treatment of IS and to quantify the subjective experience of scoliosis and surgery and compare with established objective measurements of scoliosis deformity.

The findings of this study provide an original contribution to knowledge in ankylosing spondylitis (AS) and have important implications for eiThancing clinical practice. The results demonstrate the existence and significance of associations between disease and psychological status in AS, and also demonstrate the value of using longitudinal, repeated measures approach to study this long-term condition. This study is also the first to demonstrate the value of using a mixed methods approach to investigate this issue in AS. Although existing literature on prospective longitudinal cohort studies in AS is very limited (other than for studies which involve clinical trials of medications and other interventions), this project demonstrates the feasibility of sustaining such a study over an 18-month period and of recruiting large numbers of participants to both the quantitative and qualitative phases. The results are based upon a hospital-ascertained cohort of 89 adults. Both the quantitative and qualitative phases produced important new findings: 1. In this cohort, mean BASMI, BASFI and BASDAI scores remained consistent throughout the 18-month period. People with BASDAI scores higher than 4 at the beginning of the study continued to score higher than 4 throughout. 2. BASMI, BASFI and BASDAI scores correlated significantly with anxiety, depression and internality scores, but not with levels of belief in chance or powerful others, throughout the study. This demonstrates that AS disease status is closely linked to some, but not all, psychological measures. 3. There was no effect of co-existent psoriasis or iritis on either disease or psychological status, but BASMI and BASFI (but not BASDAI) scores were significantly related to age. 4. Factors which appear to influence the associations between disease and psychological status are highly complex, often differing between individuals, and usually determined by other co-morbidities and life circumstances besides AS. These results suggest that the major implication for clinical practice would be the development of a more comprehensive and integrated assessment framework for AS set within the context of a biopsychosocial model. Envisaged would be a major programme of work to critically assess and validate potential components of such a framework with the aim of determining efficacy, feasibility and acceptability of such an approach.

Neural tube defects are a set of developmental malformations which can be highly debilitating, with limited treatment available. Mouse mutants exhibiting neural tube defects are studied to identify processes promoting proper neural tube closure, and potential points of intervention for future therapies. This thesis characterises the mouse mutant hitchhiker (hhkr), a hypomorphic allele of Tulp3 which presents with neural tube defects and polydactyly. The spina bifida and exencephaly observed in hhkr mutants are demonstrated to be consequences of failure of neural tube closure, and excessive proliferation is identified in the hindbrain neuroepithelium of mutant embryos. Intriguingly, increases apoptosis was reported for the Tulp3tmlJng mutant (lkeda et aI., 2001), and this increase is not conserved in Tulp3hhkr. Further support is provided for the role of Tulp3 as a negative regulator of Sonic hedgehog (Shh) signalling, confirming such a role in the limb, while preliminary data from genetic interaction studies between hhkr and Tectonic-/- are presented to suggest Tulp3 may exert a positive influence on Shh signalling in cranial regions. The molecular function of the Tulp3 protein is investigated, revealing an interaction between Tulp3 and Alx1, a transcription factor involved in skeletal patterning. An interaction between Tulp3 and Trim71, an E3 ubiquitin ligase is also demonstrated and supported by the eo- localisation of the proteins in transfected cells. Tulp3 is shown to be ubiquitinated in vivo, although this modification does not appear to be dependent on Trim7!. This thesis provides evidence that Tulp3 is likely to be involved in diverse protein-protein interactions around the cell, and some of these interactions may be crucial in promoting the proper closure of the neural tube.

Σκοπός: Ο νεοεννευρωμένος μεσοσπονδύλιος δίσκος (ΜΔ), κλινικά θεωρείται μια σημαντική πηγή πόνου στους ασθενείς με οσφυαλγία. Η ταυτοποίηση των νεοσχηματιζόμενων νεύρων, που εκφράζουν τους υποδοχείς των νευροτροφινών, στην εκφύλιση του ΜΔ, σπονδυλικών επιπέδων που αναφέρονται ως πηγή δισκογενή πόνου και η συσχέτισή τους με το ενδοκανναβινοειδές σύστημα αναλγησίας, αποτελεί το αντικείμενο αυτής της μελέτης. Υλικό και μέθοδος: Συλλέχθηκαν 50 ανθρώπινοι ΜΔ ή τμήματα αυτών, μετά από δισκεκτομή σε χειρουργεία κήλης και εκφυλιστικών παθήσεων της Οσφυϊκής Μοίρας της Σπονδυλικής Στήλης (ΟΜΣΣ). Εφαρμόσθηκε ανοσοϊστοχημεία για την ανίχνευση νευρικών ινών, στους ΜΔ, αφού διαπιστώθηκε η εκφύλισή τους. Συσχετίσθηκε η ανάπτυξη νευρικών ινών των εκφυλισμένων ΜΔ με την έκφραση των υψηλής και χαμηλής συγγένειας υποδοχέων των νευροτροφινών, TrkA, TrkB, TrkC και p75, αντίστοιχα. Στα περιστατικά με ανάπτυξη νευρικών ινών ανιχνεύθηκε ανοσοϊστοχημικά η έκφραση του υποδοχέα CB1 των ενδοκανναβινοειδών. Αποτελέσματα: Η εκτεταμένη νεύρωση στον ΜΔ που φέρεται στον έσω ινώδη δακτύλιο (ΙΔ) και μπορεί να φτάνει μέχρι τον πηκτοειδή πυρήνα (ΠΠ), φαίνεται να είναι εξαρτώμενη από τους υποδοχείς των νευροτροφινών TrkA, TrkB, TrkC και p75. Μικρά αιμοφόρα αγγεία συνοδεύουν τις νευρικές ίνες στα σπονδυλικά επίπεδα του ΜΔ που δίνουν δισκογενή πόνο και εκφράζουν επίσης τους υποδοχείς των νευροτροφινών. Ο CB1 υποδοχέας των ενδοκανναβινοειδών ταυτοποιήθηκε σε όλα τα περιστατικά που εμφάνιζαν ανάπτυξη νευρικών ινών. Βρέθηκε να εκφράζεται στα αγγεία που συνοδεύουν τις νευρικές ίνες και σε μερικά νεύρα. Δεν ταυτοποιήθηκε ανάπτυξη νευρικών ινών σε εκφυλισμένους ΜΔ σπονδυλικών επιπέδων που δεν αποτελούσαν αιτία δισκογενούς οσφυαλγίας. Συμπέρασμα: Η αλγοϊδιοδεκτική ανάπτυξη νευρικών ινών στον ΜΔ συνδέεται με την έκφραση των υποδοχέων των νευροτροφινών στις νεοσχηματιζόμενες νευρικές ίνες, δείχνοντας ότι όχι μόνο ο NGF, αλλά και άλλες νευροτροφίνες εμπλέκονται στην ανάπτυξη νευρικών ινών. Η έκφραση του CB1 υποδοχέα των ενδοκανναβινοειδών αποτελεί πεδίο έρευνας για τη θεραπεία του δισκογενή πόνου. / The current study was designed in order to detect immunohistochemically the expression of cannabinoid CB1 receptor and high and low affinity neurotrophin receptors TrkA, TrkB, TrkC and p75 respectively in human painful intervertebral disc (IVD). Previous data have shown an association between nerve ingrowth in human degenerated IVD and patient’s experience of pain. The nociceptive nerve ingrowth into painful IVD is causally linked with Nerve Growth Factor (NGF) production by blood vessels growing into the IVD. NGF causes further innervation and is involved in processes that result in an inflammatory hyperalgesia. There is evidence that cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of proinflammatory neuroimmune processes. A total of 50 IVD specimens after lumbar discectomy were included in the present study. The degeneration of IVD was identified and connected with clinical background of patients. An immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and specific antibodies against CB1 and neurotrophin receptors (TrkA, TrkB, TrkC and p75) were used. Immunohistochemistry was also performed to detect neural and vascular markers: tubulin (βIII) and CD31. Neurotrophin receptors (TrkA, TrkB, TrkC and p75) expression was detected only in innervated degenerated IVD. Blood microvessels accompanied nerve fibers growing into IVD expressed also neurotrophin receptors. Furthermore, in some nerve fibers CB1 cannabinoid receptor expression was identified. Interestingly, CB1 receptor expression was detected in the majority of vessels accompanying nerve fibers. Nociceptive nerve ingrowth into degenerated IVD was strongly associated with all neurotrophin receptors expression indicating that not only NGF but also and others neurotrophins are involved in nerve ingrowth. Expression of CB1 cannabinoid receptor in innervated IVD suggests a possible therapeutic site of analgesic action. Additional studies would be necessary to clarify the functional role of CB1 cannabinoid receptor in degenerated intervertebral disc.

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The Intervertebral disc (IVD), comprised of two distinct regions, namely the fibrous annulus fibrosus (AF) and the gelatinous nucleus pulposus (NP), is a fibrocartilage pad located between adjoining vertebrae of the spine. The function of the IVD is to provide stability to the spine, while maintaining movement. IVD degeneration, also known as degenerative disc disease (DDD), is the process whereby the IVD tissue degrades, resulting in loss of function to the disc. Low back pain (LBP) is associated with the degeneration of the IVD, making it important to investigate the pathogenesis of DDD, as this could lead to novel therapies for the prevention and/or treatment of LBP. Mechanical stimuli (MS) are known to be important for IVD cell matrix homeostasis, with cells of the AF and NP responding to physiological forces with a trend towards increased matrix anabolism, while non-physiological forces lead to matrix catabolism. Furthermore, recent evidence suggests that IVD cells derived from degenerate tissue may have lost their ability to respond to physiological MS in the 'normal' anabolic manner, potentially leading to the progression of DDD. It is therefore important to investigate the response of IVD cells derived from both non-degenerate and degenerate tissue to MS, to ascertain whether there is a difference with degeneration. If the response is found to be altered with degeneration, then elucidation of the potentially altered mechanotransduction pathway utilised by degenerate cells could lead to the discovery of novel therapeutic targets for the treatment of DDD. To date, the majority of IVD MS studies have concentrated on the response of NP cells to hydrostatic pressure, with only a limited number of AF studies available. Thus, the first aim of this PhD was to investigate the response of human AF cells derived from non-degenerate and degenerate IVDs to the physiologically relevant mechanical stimulus of cyclic tensile strain (CTS), to ascertain whether the response (regulation of matrix protein and matrix degrading enzyme gene expression) was frequency-dependent or altered with IVD degeneration. Using an in vitro mechanical loading system (Flexcell® Tension Plus™ system, Flexcell International) capable of delivering a CTS of 10% strain, 0.33Hz or 1.0Hz for 20 minutes, the response of AF cells derived from non-degenerate IVDs was found to be frequency-dependent (reduced catabolism at 1.0Hz, with decreased MMP -3 and ADAM-TS -4 gene expression; and catabolic at 0.33Hz, with decreased types I and II collagen and increased MMP -9 gene expression). Furthermore, the response of AF cells to 1.0Hz CTS was shown to be altered with IVD degeneration, depicted by a switch from reduced catabolism (decreased MMP -3 and ADAM-TS -4) in non-degenerate AF cells, to reduced anabolism (decreased aggrecan and type I collagen gene expression) in degenerate AF cells. Subsequently, the second aim of the PhD was to attempt to elucidate the mechanotransduction pathways operating in human AF cells derived from non-degenerate and degenerate IVDs, to ascertain whether the mechanotransduction pathway was altered with IVD degeneration. An identical mechanical stimulation regime was used (1.0Hz CTS) in parallel with functional inhibitors against the cytokines interleukin (IL) -1 and -4, and the cell surface receptors, RGD-recognising integrins. Additionally, the involvement of the cytokine associated transcription factors, nuclear factor kappa beta (NFκB) and signal transducer and activator of transcription (STAT) -6, as well as the integrin-associated kinase, focal adhesion kinase (FAK) was investigated in 1.0Hz CTS-treated non-degenerate AF cells. The response to 1.0Hz CTS (reduced catabolism) of AF cells derived from non-degenerate IVDs occurred in an IL -1, IL -4 and RGD-recognising integrin-dependent manner, with FAK being phosphorylated. Of significant interest, the altered response of AF cells derived from degenerate IVDs to 1.0Hz CTS (reduced anabolism) occurred independently of either cytokine and independently of RGD-recognising integrins, suggesting an altered mechanotransduction pathway in operation and warranting further investigation.

Πρόκειται για μια προοπτικη, συγκριτική, τυχαιοποιημένη, κλινική και ακτινολογική μελέτη της εξέλιξης της πώρωσης οπισθοπλάγιας σπονδυλοδεσίας με τη χρήση είτε αυτόλογου μοσχευματός είτε κοραλλιογενους υδροξυαπατίτη είτε μιγματος των δύο. Μελετήθηκαν 3 ομάδες ασθενών Α, Β και Γ με 18, 19 και 20 ασθενείς αντιστοιχά. Στην πρώτη ομάδα χρησιμοποιήθηκε μόνο αυτόλογο μόσχευμα, στη δεύτερη κοραλλιογενής υδροξυαπατίτης και αυτόλογο μόσχευμα και στην τρίτη ομάδα μόνο κοραλλιογενής υδροξυαπατίτης. Η εκτίμηση ήταν κλινική και ακτινολογική. Διαπιστώθηκε ότι η ενσωμάτωση του κοραλλιογενούς υδροξυαππατίτη χρειάζεται επααρκή αποφλοιωμένη οστική επιφάνεια και η τοποθετηση του στα οπισθοπλάγια σπονδυλικά στοιχεία δεν οδηγεί σε σπονδυλοδεσία. Όμως η τοποθετησή του κοραλλιογενούς υδροξυαπατίτη στα οπίσθια σπονδυλικά στοιχεία οδηγεί σε σπονδυλοδεσία στον αναμενόμενο χρόνο. / This prospective longitudinal randomized clinical and radiological study compared the evolution of instrumented posterolateral lumbar and lumbosacral fusion using either coralline hydroxyapatite or iliac bone graft or both. Three comparable groups A, B, and C were evaluated. In group A only autograft was used. In group B autograft and coralline hydroxyapatite were used and in group C only coralline hydroxyapatite was used as graft extender. The goups were evaluated clinically and radiologicalý It was found that coralline hydroxyapatite applied in the poserolateral spinal elements does not leed to a spinal fusion because of lack of bleeding osseous surfaces. However if coralline hydroxyapatite is applied in the laminae of the posterior spine it leeds to spinal fusion within the expected time.